Chronic myeloid leukemia in chronic phase (CML-CP) is initiated by t(9;22) encoding for p210BCR-ABL1 tyrosine kinase that transforms hematopoietic stem cells (HSCs). CML-CP is leukemia stem cells (LSCs) - derived disease, but deregulated growth of LSCs-derived leukemia progenitor cells (LPCs) leads to the manifestation of the disease. CML-CP may progress to more advanced accelerated phase (CML-AP), and subsequently to a very aggressive blast phase (CML-BP). Most CML-CP patients are currently treated with tyrosine kinase inhibitors (TKIs) such as imatinib, dasatinib and nilotinib. However, it is unlikely that TKIs will cure CML patients due to the presence of TKI- refractory cells (e.g, quiescent LSCs) and TKI-resistant cells (e.g., proliferating LSCs/LPCs carrying BCR- ABL1 kinase T315I mutant). In addition, population studies revealed that overall only 51% of CML-CP patients respond favorably to TKI treatment. Therefore, novel treatment modalities are needed to eradicate TKI- refractory/resistant CML cells in the responding patients and also to treat patients who do not respond favorably to TKIs. To cure CML these strategies should simultaneously target two fundamentally different leukemia cell populations: TKI-refractory quiescent LSCs and TKI-resistant/refractory proliferating LSCs/LPCs. We found that CML LSCs and LPCs, including quiescent LSCs accumulate 2-4 times more reactive oxygen species (ROS)-induced DNA double strand breaks (DSBs) than normal counterparts (Cramer et al., Cancer Res., 2008; Nieborowska-Skorska et al., Blood, 2012; Bolton-Gillespie et al., Blood, 2013). DSBs are the most lethal DNA lesions. We reported that CML cells can tolerate high numbers of DSBs because two major repair mechanisms, homologous recombination repair (HRR) and non-homologous end-joining (NHEJ) are hyper- activated (Slupianek et al., Mol. Cell, 2001; Oncogene, 2005; DNA Repair, 2006; Cancer Res., 2011; Blood, 2011; Nowicki et al., Blood, 2005). CML cells are addicted to these pathways to survive pro-apoptotic challenge from high numbers of lethal DSBs. However, there are critical differences between DSB repair in normal and CML cells. Proliferating LSCs/LPCs employ RAD52-dependent HRR, in contrast to BRCA1-mediated HRR in normal counterparts. Quiescent LSCs use PARP1-mediated NHEJ instead of DNA-PKcs -dependent NHEJ, which is predominant in normal quiescent HSCs. We will explore these differences to target leukemia-specific DNA repair mechanisms simultaneously in quiescent LSCs and proliferating LSCs/LPCs to achieve dual synthetic lethality, with negligible effect on normal cells and tissues. According to our best knowledge the concept of dual synthetic lethality was not tested before. Dual synthetic lethality will be induced in TKI-treated CML-CP/AP cells by simultaneous targeting of RAD52 and PARP1 using recently identified candidate small molecule inhibitors interrupting key functions of RAD52 and PARP1: RAD52 DNA binding activity and stimulation of PARP1 by histone 4. In addition, using CML-CP -like transgenic mice, Rad52-/-Parp1-/- double knockout mice, mutagenic approach, peptide aptamers, and CML-CP/AP primary cells we will determine if other RAD52 and/or PARP1 activities could be targeted to trigger more efficient dual synthetic lethality simultaneously in TKI-refractory quiescent LSCs and TKI-resistant proliferating LSCs/LPCs. Our long-term plan is to run a clinical trial testing the possibility to eradicate CML-CP/AP by induction of dual synthetic lethality in TKI-treated patients.